As in previous years, a major focus of this project has been detailed longitudinal study of the behavioral and biological consequences of differential early social rearing, most notably comparing rhesus monkey infants reared by their biological mothers and others with same-age infants for their first 6-7 months of life (MR), with monkeys separated from their mothers at birth, hand-reared in the labs neonatal nursery for their first month and then raised in small groups of same-age peers for their next 6 months or housed in individual cages containing an inanimate surrogate mother and given 2 hours of daily interaction with like-reared peers (NR). At 7-8 months of age, MR and NR infants are all moved into one large pen, where they all live together until puberty. Thus, the differential social rearing occurs only for the first 7-8 months; thereafter MR and NR all share the same physical and social environment. We previously demonstrated that NR monkeys cling more, play less, tend to be more impulsive and aggressive, and exhibit much greater behavioral and biological disruption during and immediately following short-term social separation at 6 months of age than MR monkeys, and they also exhibit deficits in serotonin metabolism (as indexed by chronically low values of CSF 5-HIAA).. Additionally, they have significantly lower levels of 5-HTT binding throughout many brain regions than do MR subjects. Many of these differences between MR and NR monkeys persist throughout the childhood years in the absence of experimental interventions. For example, this past year we published data reporting that adult female NR monkeys had shorter lymphocyte telomeres than their MR counterparts, We have also found that NR monkeys required greater veterinary attention with respect to treatment for G-I disorders, infections, and wounds than MR monkeys during adolescence and early adulthood Another major focus of recent research for this project has involved characterizing interactions between differential early social rearing and polymorphisms in several candidate genes (G X E interactions), most notably the 5HTTLPR gene. During the past two years we expanded the range of outcomes for which G x E interactions involving the 5-HTTLPR polymorphism and early rearing condition differences appear, including social play, and behavioral reactions to a variety of social stressors, and in epigenetic regulation of brain activity. In addition, we recently reported significant G x E interactions between early MR vs.NR rearing and polymorphisms for several other candidate genes, including DRD1, neuropeptide Y, mu opioid (OPRMI), BDNF, NOS-1, and a SNP in the glucocorticoid gene, with outcome measures including aggression, play behavior, social buffering, behavioral and HPA reaction to an unfamiliar conspecific, naloxone treatment, alcohol consumption, and plasma BDNF concentrations. In virtually every case a similar pattern has been observed: The less efficient (transcription-wise) allele was associated with a negative outcome among NR reared monkeys but a neutral or, in some cases, even an optimal outcome for MR reared subjects carrying that same less efficient allele, suggesting an overall buffering effect of MR rearing for individuals carrying these so-called risk alleles. Additionally, we recently published the results of two sets of studies investigating the effects of differences in early social rearing (MR vs. NR) on genome-wide patterns of mRNA expression in leukocytes, and on methylation patterns in prefrontal cortex and in T-cell lymphocytes. Our research involving mRNA expression, carried out in collaboration with Steven Cole and James Heckman, examined expression patterns in differentially reared 4-month-old infants. In all, 521 different genes were significantly more expressed in MR infants than in SPR infants, whereas the reverse was the case for another 717 genes. In general, NR infants showedenhanced expression in genes involved in inflammation, T-lymphocyte activation and cell proliferation, and suppression of antiviral and antibacterial responses. Since that initial study we have completed a prospective longitudinal study in which differentially reared subjects are being sampled at 14 days, 30 days, 6-7 months, and every 3 months thereafter until they reach puberty. Data analyzed to date have revealed that the above rearing condition differences in genome-wide patterns of mRNA expression in leukocytes persist throughout development in the absence of any changes in the social environment but change dramatically whenever the social environment is altered during the juvenile years. These new findings are currently being prepared for publication. The other set of studies, carried out in collaboration with Moshe Szyf and his lab at McGill University, involved genome-wide analyses of methylation patterns in differentially reared monkeys when they were adults. The initial study compared such patterns in prefrontal cortex tissue and T-cell lymphocytes obtained from 8-year-old monkeys differentially reared for their initial 6-7 monthsand thereafter maintained under identical conditions until adulthood. These analyses revealed that (a) more than 4,400 genes were differentially methylated in both PFC and lymphocytes, (b) although there was considerable tissue specificity, approximately 25% of the affected genes were identical in both PFC and lymphocytes, and (c) in both PFC and lymphocytes methylated promoters tended to cluster both by chromosomal region and gene function. This past year we completed a prospective longitudinal study of genome-wide methylation patterns in lymphocytes, collecting samples from exactly the same MR and SPR monkeys at exactly the same time points as in the afore-mentioned study of mRNA expression. Finding published this past year suggest that, at least in lymphocytes, extensive rearing conditions are present within the first month of life but can at least be significantly reversed following a social environmental intervention utilizing foster grandparents. Recently, we also published the results of a long-term longitudinal study detailing genome-wide epigenetic changes in some of these MR and PR monkeys over their first 2 years of life. We found dramatic changes in methylation patterns in MR monkeys from infancy to 6 months in both males and females, affecting wide swaths of the genome, but sex differences in infancy were largely reversed prior to weaning. These differences continued after weaning, albeit with some attenuation, but increased again by 2 years of age. Both sexes of NR monkeys exhibited very different developmental trajectories in infancy, showing many of the same genome-wide patterns seen in post-weaning MP monkeys, with parallel sex differences. In sum, genome-wide patterns of methylation in lymphocytes were highly dynamic throughout pre-pubertal development and varied dramatically as a function of setting, sex and early social rearing history. Finally, in another collaboration with the Szyf lab, we published a paper that examined the epigenetic consequences of being high vs. low-ranking in established social groups of adult female monkeys and their offspring, whose relative social dominance status matched that of their mothers. It appeared that the cross-generational transmission of social status was mediated by, at least in part, the placenta, in that the genome-wide pattern of methylation in tissues collected from placentas immediately after birth differed dramatically between offspring of high- vs. low-ranking females, and not only did the order of magnitude of these differences match that of the above-mentioned rearing condition differences but also many of the same sets of genes were involved, suggesting the existence of a subset of adversity genes sensitive to a range of early social adversities.